In the prior art, examples of methods for preparation of industrially important optically active alcohols including ethyl (S)-4-chloro-3-hydroxybutyrate with the use of wild type whole cell biocatalysts isolated from variant, sources like Geotrichum candidum (Sundby, E. et al. Journal of Molecular Catalysis B: Enzymatic 2003, 21, 63-66), Candida parapsilosis (Kaliaperumal, T. et al. Journal of Industrial Microbiology & Biotechnology 2010, 37, 159-165), Candida magnoliae (Yasohara, Y. et al. Applied Microbiology and Biotechnology 1999, 51, 847-851), Cylindrocarpon sclerotigenum (Saratani, Y. et al. Bioscience, Biotechnology, and Biochemistry 2001, 65, 1676-1679), Kluveromyces lactis (Yamamoto, H., et al. Bioscience, Biotechnology, and Biochemistry 2002, 66, 1775-1778), Kluyveromyces aestuarii (Yamamoto, H. et al. Bioscience, Biotechnology, and Biochemistry 2004, 68, 638-649), Aureobasidium pullulans (He, J. Y. et al. Process Biochemistry 2006, 41, 244-249), Pichia stipitis (Ye, Q. et al. Biotechnology Letters 2009, 31, 537-542) and Streptomyces coelicolor (Wang, L. J. et al. Bioresource Technology 2011, 102, 7023-7028) have been described. Further, methods for producing ethyl (S)-4-chloro-3-hydroxybutyrate with the use of wild type whole cell biocatalysts have been disclosed in U.S. Patent Ser. No. 99/5891685; U.S. Patent Ser. No. 97/5700670; U.S. Patent Ser. No. 96/5559030; U.S. Patent Ser. No. 95/5413921; U.S. Patent Ser. No. 90/4933282 and U.S. Patent Ser. No. 87/4710468. Although, the methods that describe use of wild type microorganism for reduction of carbonyl group to corresponding alcohol exist, these suffer from drawbacks, such as lower efficiency, lower substrate concentration, lower optical purity, etc. Therefore, it is impractical to synthesize industrially important optically active alcohols including ethyl (S)-4-chloro-3-hydroxybutyrate using wild type natural whole cell biocatalysts.
Further known methods to improve optical purity of industrially important optically active alcohols including ethyl (S)-4-chloro-3-hydroxybutyrate, include the use of an enzyme purified from the native source, like Candida magnoliae (Wada, M. et al. Bioscience, Biotechnology, and Biochemistry 1998, 62, 280-285), Sporobolomyces salmonicolor (Kita, K. et al. Journal of Molecular Catalysis B: Enzymatic 1999, 6, 305-313), Kluyveromyces lactis (Yamamoto, H. et al. Bioscience, Biotechnology, and Biochemistry 2002, 66, 1775-1778), Kluyveromyces aestuarii (Yamamoto, H. et al. Bioscience, Biotechnology, and Biochemistry 2004, 68, 638-649), Pichia stipitis (Ye, Q., et al. Bioresource Technology 2009, 100, 6022-6027) and Streptomyces coelicolor (Wang, L. J. et al. Bioresource Technology 2011, 102, 7023-7028).
When a purified enzyme or transformant having carbonyl reductase activity reduces carbonyl group of ketones including ethyl 4-chloro-3-oxobutyrate, it requires a coenzyme, nicotinamide adenine dinucleotide, reduced (NADH) or nicotinamide adenine dinucleotide phosphate, reduced (NADPH) for preparation of industrially important optically active alcohols including ethyl (S)-4-chloro-3-hydroxybutyrate. As the reaction proceeds, coenzyme is converted into nicotinamide adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide adenine dinucleotide phosphate (NADP). In the absence of cofactor regenerating system, stoichiometric amount of expansive cofactor is required. However, when the reaction is done in presence of a cofactor regenerating system, the amount of an expansive coenzyme is greatly reduced. A cofactor regenerating system typically consists of an enzyme which in presence of its substrate converts nicotinamide adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide adenine dinucleotide phosphate (NADP) to nicotinamide adenine dinucleotide, reduced (NADH) or nicotinamide adenine dinucleotide phosphate, reduced (NADPH). Coenzyme regeneration ability can be fulfilled either by the use of purified enzyme (Yasohara, Y. et al. Bioscience, Biotechnology, and Biochemistry 2000, 64, 1430-1436; Kaluzna, I. A. et al. Journal of the American Chemical Society 2004, 126, 12827-12832; Zhu, D. et al. The Journal of Organic Chemistry 2006, 71, 4202-4205; Ye, Q. et al. Biotechnology Letters 2009, 31, 537-542), or a transformant having coenzyme regeneration ability in the cytoplasm (Xu, Z. et al. Applied Microbiology and Biotechnology 2006, 70, 40-46; Zhang, J. et al. Chemical Communications 2006, 398-400).
Methods such as in vitro enzyme evolution using gene shuffling technologies have been employed to improve the activity of a ketoreductase that asymmetrically reduces ethyl 4-chloro-3-oxobutyrate by about 13-fold and glucose dehydrogenase that recycles cofactor nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide phosphate, reduced (NADPH) using glucose a substrate by about 7-fold compared to corresponding wild type enzyme (Steve K. Ma et al. Green Chemistry 2010, 12, 81-86; U.S. patent Ser. No. 10/002,8972; U.S. patent Ser. No. 10/781,6111).
However, the use of an isolated enzyme requires additional steps, such as isolation, purification and stabilization of enzyme, which adds to the cost and makes the overall process for production of optically active alcohols economically unattractive. In addition, inhibition of enzyme by substrate and/or product can sometimes occur. The use of two enzymes, one for reduction of carbonyl compound and other for cofactor recycling along with two substrates, one for each enzyme leads to complex overall kinetics of the reaction. Put together, these factors make the use of isolated enzymes less attractive compared to use of whole cells in the preparation of enantiomerically enriched alcohols including ethyl (S′)-4-chloro-3-hydroxybutyrate.
To overcome the problem of low efficiency associated with wild type strains, the gene encoding the carbonyl reductase activity can be deduced and overexpressed in a host cell. Further improvement in efficiency of whole cell biocatalyst for production of enantiomerically enriched alcohols including ethyl (S)-4-chloro-3-hydroxybutyrate is achieved by coexpressing both carbonyl reductase and coenzyme regeneration ability in the cytoplasm of same host (Kizaki, N. et al. Applied Microbiology and Biotechnology 2001, 55, 590-595, Kataoka, M. et al. Enzyme and Microbial Technology 2006, 38, 944-951; Ye, Q. et al. Bioresource Technology 2010, 101, 6761-6767).
However, the method that use either a transformant wherein carbonyl reductase ability is present or a transformant wherein both carbonyl reductase and coenzyme regenerating ability is present in the cytoplasm of cells suffers from drawbacks such as low efficiency due to barrier imposed by plasma membrane on substrate uptake and product efflux, complex kinetics of the overall process, etc.
Consequently, the current methods for the production of optically pure ethyl (S)-4-chloro-3-hydroxybutyrate and other optically enriched alcohols suffer from drawback such as low efficiency, low productivity, etc., which therefore results in increased cost of production.
The art of expressing a protein including enzymes on surface of cells is well known (Lee, S. Y. et al. Trends in Biotechnology 2003, 21, 45-52) and has been used in a wide range of biotechnological and industrial applications like whole-cell biocatalyst for bioconversion (Shimazu, M. et al. Protein. Biotechnology Progress 2001, 17, 76-80; Shimazu, M. et al. Biotechnology and Bioengineering 2001, 76, 318-324), bioadsorbent for the removal of harmful chemicals and heavy metals (Bae, W. et al. Biotechnology and Bioengineering 2000, 70, 518-524; Bae, W. et al. J Inorg Biochem 2002, 88, 223-227; Sousa, C. et al. J Bacteriol. 1998, 180, 2280-2284; Xu, Z. et al. Appl Environ Microbiol 1999, 65, 5142-5147), screening of human antibodies libraries (Chao, G. et al. Nat. Protocols 2006, 1, 755-768), mutation detection (Aoki, T. et al. Analytical Biochemistry 2002, 300, 103-106), biosensor development by anchoring enzymes, receptors or other signal-sensitive components (Dhillon, J. K. et al. Letters in Applied Microbiology 1999, 28, 350-354; Shibasaki, S. et al. Applied Microbiology and Biotechnology 2001, 57, 702-707; Shibasaki, S. S. et al. Applied Microbiology and Biotechnology 2001, 57, 528-533).
More recently, a method for simultaneous display of a target protein including dehydrogenase but not carbonyl reductase and glucose dehydrogenase has been disclosed in U.S. patent Ser. No. 11/789,7366. Usually the cell surface proteins or their truncated form (carrier proteins) fused with the target peptide or protein (passenger protein) are used to display the protein on the surface. Gram (−) ve (Escherichia coli), gram (+) ve (Bacillus subtilis, Staphylococcus strains) bacteria and yeast (Saccharomyces cerevisiae, Pichia pastoris) have been explored for display of heterologous protein expression on the surface. Rigid structure of the cell wall of gram (+) ve bacteria makes it a suitable host, however, the gram (−) ve Escherichia coli has been the most popular and much explored for the cell surface display. Integral proteins of Escherichia coli, such as LamB, FhuA, and the porins OmpA, OmpC and OmpX, which give structural rigidity to outer membrane has been extensively used for insertion of short amino acid sequence (up to 60 amino acid) in extracellular loop and display it on the cell surface. Outer membrane lipoproteins are anchored in the membrane by a small lipid modified amino terminal. The first lipoprotein based cell surface display was Lpp-OmpA chimera consisting of the 20 amino acid signal sequence and first nine N-terminal residues of the mature Escherichia coli lipoprotein, and the residues 46-159 of the Escherichia coli outer membrane protein A (OmpA) were fused to the N-terminal of the passenger protein.